DNA mismatch repair (MMR) plays a number of critical roles in eukaryotic cells including: 1) suppression of mutations that result from misincorportation errors during DNA replication as well as chemical damage to DNA and DNA precursors; 2) repair of mispaired bases in recombination intermediates; 3) preventing recombination between divergent DNA sequences; and 4) signaling the presence of DNA damage resulting in cellular responses such as cell cycle arrest and cell death. As a consequence of these cellular roles, MMR defects increase the spontaneous mutation rate and the rate of altered recombination events resulting in a characteristic genome instability signature as well as resulting in increased resistance to killing by some DNA damaging agents. Understanding the mechanism of MMR will impact human health for a number of reasons: 1) Hereditary non-polyposis colon cancer is due to inherited defects in MMR and many sporadic cancers appear MMR defective, yet the genetic consequences of MMR defects are not fully understood; and, 2) many chemotherapy agents damage DNA and MMR defects can result in resistance to some of these agents so understanding MMR could lead to improvements in the efficacy of these agents as well as ways to circumvent MMR defect-mediated resistance. The goal of this proposal is to use Saccharomyces cerevisiae to study the biochemical and genetic mechanisms of the eukaryotic MutS- and MutL-horriologue dependent MMR pathways. The following lines of experimentation will be carried out: 1) genetic studies will identify MMR genes and mutations for use in dissecting the biochemical properties of MMR proteins; 2) biochemical studies of individual MMR proteins including the Msh2-Msh3, Msh2-Msh6, Mlh1-Pms1 and Mlh1-Mlh3 complexes, RPA, PCNA, RFC, Exo1, and other proteins will be continued to determine the roles these proteins play in MMR; 3) the biochemical basis for the higher order protein complexes that function in MMR will be determined; 4) partial and complete MMR reactions will be reconstituted in vitro using purified proteins to study the mechanisms of MMR; and 5) collaborative mouse model studies will be continued to extend insights from studies with S. cerevisiae to mammalian systems. The ultimate goal of these experiments is to reconstitute MMR with purified proteins and determine the mechanisms of these reactions. A key feature of these studies is the use of S. cerevisiae to explore questions raised by the genetics of human cancer susceptibility, and collaborative mouse studies to explore the broader implications of results developed in S. cerevisiae. As a consequence, it is anticipated that these studies will provide genetic and biochemical insights that can be applied to the study of the genetics of human cancer susceptibility.